(1) Field of the Invention
This invention relates to nucleic acid sequences of the blueberry red ringspot virus and, more particularly, to the nucleic acid sequence of the virus, the identification of promoters, and the use of the promoters in the expression of recombinant genes in transgenic plants, including tissue-specific expression in plants. The invention also relates to sequences of the blueberry red ringspot virus useful in the diagnosis of disease in plants, and related methods thereof.
(2) Description of the Related Art
Recombinant viral promoters can be used to direct the expression of operably linked heterologous genes. Such expression can occur in a transgenic environment. For expression of transgenes in plants, a promoter from Cauliflower Mosaic Virus (CaMV) is widely used, for example as disclosed in U.S. Pat. No. 6,255,560 to Fraley et al. Expression of transgenes in plants under the control of a CaMV promoter tends to be at high levels and show little tissue- or cell-type specificity. This virus is unusual, in that it appears to comprise only two promoters. One promoter appears to control the transcription of the entire viral genome into an RNA copy. This promoter (the “35S” promoter) is a tandem repeat of an approximately 350 base pair sequence. Within this sequence are domains involved with tissue specific expression of genes expressed from the promoter (Odell, J. T., Nagy, F. and Chua, N. H. (1985) Identification of DNA sequences required for the activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810-812; Benfey, P. N. and Chua, N. H. (1990) The Cauliflower Mosaic Virus 35S promoter: combinatorial regulation of transcription in plants. Science 250: 959-966; Daubert, S. D., et al., (1984) Expression of Disease Symptoms in CaMV Genomic Hybrids. Journal of Molecular and Applied Genetics 202: 1043-1045; Dixon, L. K., et al., (1983) Mutagenesis of Cauliflower Mosaic Virus. Gene 25: 189-199; Mesnard, J., et al. (1990) The Cauliflower Mosaic Virus Gene III product is a non-sequence specific DNA binding protein. Virology 174: 622-624; Pfeiffer, P. and Hohn, T. (1983) Involvement of reverse transcriptase in the replication of cauliflower mosaic virus. Cell 33: 781-789; Takatsuji, H., et al. (1992) Cauliflower Mosaic Virus reverse transcriptase—activation by proteolytic processing and functional alteration by terminal deletion. Journal of Biological Chemistry 267: 11579-11585; Thomas, C. L. et al. (1992) A mutation in Cauliflower Mosaic Virus Gene I interferes with virus movement but not with virus replication. Virology 74: 1141-1148). This promoter has proven useful for directing the expression of heterologous genes in transgenic plants.
Because transgenes under CaMV promoter control may not be suitable for all uses, there is a need for the identification and characterization of more plant virus promoters. For example, there is the need for tissue specific promoters that can direct expression of an operably linked gene to a subset of tissues within a transgenic plant. Furthermore, there is a need for a strategy for identifying putative promoters, and a further need to demonstrate the operability of a putative promoter in a transgenic environment.
The blueberry red ringspot virus is a virus with a limited host range: it is believed to infect only blueberry plants. Disease symptoms are observed primarily in the months of July, August and September, and comprise red spots primarily on upper leaf surfaces (Hutchinson, M. T. (1950) Can you recognize the symptoms of stunt disease? Proceedings 19th Annual Blueberry Open House 19: 9-11; Ramsdell, D. C., Kim, K. S. and Fulton, J. P. (1987) Red Ringspot of Blueberry. In: Converse, R. H. (ed.) Virus Diseases of Small Fruits. U.S. Department of Agriculture, Agr. Res. Svc., Washington, DC Handbook 631; Kim, K. S., Ramsdell, D. C., Gillett, J. M. and Fulton, J. P. (1981) Virions and substructural changes associated with blueberry red ringspot disease. Phytopathology 71: 673-678; Gillett, J. M. (1988) Physical and Chemical properties of Blueberry Red Ringspot Virus. Master's thesis, Michigan State University; Hutchinson, M. T. and Vamey, E. H. (1954) Ringspot: A virus disease of cultivated blueberry. Plant Disease Reports 38: 260-262). A sequence of a putative blueberry red ringspot virus has been published on the http World Wide Web at ncbi.nlm.nih.gov with accession numbers NC 003138 and AF404509. This sequence has a length of 8,303 base pairs. No promoters are disclosed or identified in these sequence listings. Furthermore, this sequence has several differences with the sequence of the virus of the present invention as disclosed herein.